Transplantation has emerged as the preferred method of treatment for many forms of end-stage organ failure. While short-term results have, improved long-term outcomes remain inadequate. The development of strategies to promote the long-term acceptance of allogeneic tissues without the need for chronic immunosuppression, which defines transplantation tolerance, could greatly expand the application of organ, tissue and cellular transplantation for diseases such as the hemoglobinopathies, genetic immunodeficiencies, and possibly autoimmune diseases. However, transplantation tolerance has remained an elusive goal in clinical transplantation. It has been known for many years that mixed hematopoietic chimerism induces a state of specific immunological tolerance, and in recent years it has been shown that this goal can be achieved in mouse models using clinically relevant costimulation blockade based strategies. While there are many practical issues that must be addressed for tolerance induction to become a clinical reality, we believe that the strategy must provide a means to control the existing population of donor-specific T cells in the periphery. This includes the control of memory cells that may persist in an individual as a result of previous antigen exposure. An understanding of the memory cell population is critical; both to prevent these cells from being a barrier to tolerance induction, as well as maintaining their function posttolerance induction to prevent infectious complications in transplant recipients. The specific aims of this study are to: (1) To characterize the number, distribution, phenotype and function of allospecific memory T cell subsets generated after skin or cardiac transplantation in the mouse; (2) To determine the number, subset and phenotype of allospecific memory T cells that prevents, the induction of chimerism and tolerance using the CD2B/CD40 blockade and micro-conditioning tolerance induction protocol; (3) To define strategies to tolerize allospecific CD4 and CDB memory populations by (a) blockade of alternative costimulatory pathways, or (2) manipulation of the signal 3 pathway. The results of this study will be critical for the development of safe strategies for the induction of transplantation tolerance to address the great clinical need.